Hepatocytes perform important roles in the metabolism, homeostasis of serum proteins and in the pathogenesis of various metabolic and infectious disorders. They are thus an important target for pharmacological agents. Conventional therapeutic agents that act on hepatocytes include small molecules and proteins, such as nucleoside analogues (e.g., ribavirin) and interferon used for treatment of hepatitis C [Hayden 2001]. Greater efficacy and decreased toxicity would be gained by delivering such biologically active compounds to hepatocytes in a more selective manner [Tomlinson 1987; Bijsterbosch & van Berkel 1990; Meiher et al. 1992]. Hepatocytes are also a target for a variety of anti-sense and gene therapies designed to treat liver based disorders, including: hepatitis, hypercholesterolemia and hemophilia [Chiou et al. 2001; High 2001]. However, the inability to efficiently and safely deliver nucleic acids to hepatocytes has held back the promise of nucleic acid based therapies.
One important component of any hepatocyte delivery system is a ligand that serves as a targeting “address” providing for specific and efficient delivery of therapeutic vehicles to hepatocytes [Vyas & Sihorkar 2000]. Most approaches for targeting hepatocytes have relied upon ligands that react with the asialoglycoprotein receptor (ASGPr) [Groman et al. 1994; Lee et al. 1983]. Such ligands have allowed hepatocyte targeting with small molecules, such as adenosine arabinoside, or proteins such as immunoglobulin [Rogers & Kornfeld 1971; Fiume et al. 1988]. However, there are conflicting reports concerning the utility of ASGPr ligands for targeting particles [Rensen et al. 2001; Schlepper-Schafer et al. 1986; Shimada et al. 1997; Biessen et al 1994]. A wide variety of the size limits for ASGPr-reactive particles, ranging from 10 to 70 nm, have been reported. In addition to particles and liposomes, many studies have also used ASGPr ligands for incorporation into complexes of plasmid DNA and polycations (polyplexes) [Wu et al. 2002]. The accumulated data indicate that the efficiency of hepatocyte transfection achieved with these formulations is not sufficient for clinical use.
New ligands that provide better hepatocyte specificity and potentially utilize different cellular internalization pathways would allow more flexibility in designing delivery strategies. Phage display libraries have frequently been used to identify peptide ligands that interact with specific receptors. We have previously proposed the use of T7 phage display libraries for in vivo studies because their ˜60 nm size and icosahedral shape resembles the size and shape of liposomes and DNA polyplexes.